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Abstract
An optical microfiber interferometric biosensor for the low concentration detection of sequence-specific deoxyribonucleic acid (DNA) based on signal amplification technology via oligonucleotides linked to gold nanoparticles (Au-NPs) is proposed and experimentally analyzed. The sensor uses a “sandwich” detection strategy, in which capture probe DNA (DNA-c) is immobilized on the surface of the optical microfiber interferometer, the reporter probe DNA (DNA-r) is immobilized on the surface of Au-NPs, and the DNA-c and DNA-r are hybridized to the target probe DNA (DNA-t) in a sandwich arrangement. The dynamic detection of the DNA-t was found to range from 1.0×10−15 M to 1.0×10−8 M, and the limit of detection (LOD) concentration was 1.32 fM. This sensor exhibited not only a low LOD but also excellent selectivity against mismatched DNA-t, and it can be further developed for application in various sensing platforms.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Nucleic acid detection has attracted extensive attention because the analysis of specific deoxyribonucleic acid (DNA) sequences is an important method in modern medical and healthcare diagnostics. DNA detection can promote the rapid development of gene analysis, drug research, infectious disease prevention, disease diagnosis, environmental engineering, and bioengineering effectively [1–5]. Various methods are employed to analyze and detect DNA: molecular hybridization [6,7], agarose gel electrophoresis [8], luminescence, color detection [9,10], polymerase chain reaction, and real-time fluorescence quantitative polymerase chain reaction [11]. Among these methods, molecular hybridization is especially important because it can be used to identify the target probe DNA (DNA-t) by complementary DNA strands. Electrochemistry is a common molecular hybridization method for DNA-t sequence detection because of its advantages including reliability and sensitivity [12,13]. Electrochemical techniques include using gold electrode modified by methylene blue and zirconia thin films for DNA hybridization detection [14], and label-free electrochemical transduction for COVID-19-specific viral RNA/c-DNA detection [13]. Although electrochemical techniques have many advantages, because of the large electrodes, the devices become less compact [15]. Thus, fiber optic sensors, which are promising compact photonic devices, have been employed for DNA detection in recent years because of various advantages such as flexible shape, rapid response speed, ultra-high sensitivity, real-time detection, good reliability, and intrinsic biocompatibility [16–20].
Numerous sensing mechanisms can be applied to optical fiber sensors; for example, optical fiber sensors are sensitive to the refractive index (RI) of the external environment. A long period grating (LPG) and tilted FBG (TFBG) of optical fiber sensors have been reported for biosensors because of the strong evanescent field cladding modes, which are sensitive to the RI of the surrounding environment [21–24]. To improve the evanescent field, numerous efforts have been expended continuously in recent years. Tong et al. employed a fused biconical taper to form a standard optical fiber with a diameter in sub-wavelength scale [25], and developed the new concept of “microfibers”. These microfibers boasted of advantages such as a small device size, strong evanescent field, and low transmission attenuation. Based on the characteristics of the microfiber, microfiber interferometers are a good candidate for biosensors and have rapidly developed in recent years to detect DNA, cells, and other biomolecules [26–30]. However, to further improve the detection limit of biosensors, besides the design and optimization of optical fiber sensors, signal amplification technology is another feasible method. Signal amplification methods for the detection and analysis of specific DNA sequences and other biological detection include gold nanoparticles [31–35], fluorescence [9,36], and enzyme [37]. Among various signal amplification methods, Au-NPs have attracted extensive attention and biodetection application because of their inherent stability, catalytic function, surface effect, uniform particle size, strong affinity, and good biocompatibility. Examples of Au-NPs include a novel urchin like carbon nanotube (CNT)-Au-NP nanoclusters serving as a signal amplifier [33], Au-NP functionalized surface plasmon resonance optical fiber biosensors which achieve improvement of up to three orders of magnitude [36], sensors based on horseradish peroxidase-Au-NP dual labels and lateral flow strip biosensors, which have achieved a LOD of 0.01 pM [37].
In this study, an optical microfiber interferometric biosensor for sensing DNA sequences based on a single deoxyribonucleic acid (ssDNA) functionalized on the surface was demonstrated and experimentally analyzed. As shown in Fig. 1(a), the optical microfiber interferometer was functionalized with immobilized DNA-c on the surface of the fiber, which acted as a high specificity sensor, and the DNA-c was hybridized with the DNA-t to form the double-stranded DNA (dsDNA) by base pairing. Various concentrations of DNA-t ranging from 10−17 M to 10−8 M were used for functionalization. For each concentration of DNA-t, the oligonucleotide-functionalized gold nanoparticles (Au-NPs@DNA-r) were added to hybridize with DNA-t for signal amplification. The proposed sensor is an attractive solution for the rapid and highly sensitive detection of a target in medicine, chemical, and environmental applications with low doses.
Fig. 1. (a) Schematic diagram of the sensing principle. (b) Fabrication of the optical microfiber interferometer by flame heating. (c) SEM image of the optical microfiber with a diameter of 6.6 µm. (d) Schematic diagram of the functionalized optical microfiber surface and DNA-t detection. (e) Optical fiber in the bright field of an Olympus fluorescence microscope. (f) Optical fiber without DNA-c and any fluorescent molecules. (g) Optical fiber linked DNA-c modified with 6-FAM. (h) Optical fiber modified with Cy5. (i) Atomic force microscope (AFM) image of the microfiber surface after DNA-c hybridization with DNA-t. (j) AFM image of the microfiber surface after DNA-t hybridization with DNA-r. (k) Transmission electron microscopy (TEM) micrograph of Au-NPs (l) Ultraviolet absorption spectra of Au-NPs and Au-NPs functionalized by DNA-r.
2. Experimental
2.1 Materials
All the ssDNA sequences are listed in Table 1. All sequences were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The dye (6-FAM and Cy5) used for modification of ssDNA sequences was also synthesized by Sangon Biotech. We purchased sulfuric acid (98%), hydrogen peroxide (30%) from Guangzhou Chemical Reagent Factory (Guangzhou, China). We purchased 1× Tris-EDTA buffer solution (TE, pH 7.8-8.2), ethanol (99%), and dye from Sangon Biotech, and 3-aminopropyltriethoxysilane (APTES, 99%), glutaric dialdehyde (GD, 50%), chloroauric acid (HAuCl4, 50%), and trisodium citrate (99%) were purchased from Macklin Biochemical Technology Co. Ltd. (Shanghai, China). We purchased 1× phosphate buffer saline (PBS, pH 7.2-7.4) from Solarbio Science Co. Ltd. (Beijing, China). We synthesized Au-NPs according to previous studies [38,39]. Deionized (DI) water was purified by laboratory pure water equipment from Dongguan Dow Water Treatment Equipment Engineering Co. Ltd. (Type: TS-DI-10 L/H, China), and the electrical resistivity was 15.6 MΩ.
Table 1. Single Strand DNA Sequences Used in This Study
The piranha solution was prepared by mixing sulfuric acid (98%) and hydrogen peroxide (30%) at a volume ratio of 3:1. The 5% APTES solution was obtained by dissolving ATPES (99%) in ethanol (99%). We prepared 2.5% GD by dissolving GD (50%) in 1x PBS buffer. ssDNA was dissolved in 1x TE buffer, and the DNA-c and DNA-r solutions were prepared at a concentration of 1 µM. The concentrations of the DNA-t solutions were intentionally chosen, and the 1× TE buffer attenuation range was from 10−17 M to 10−8 M.
2.2 Fabrication of the optical microfiber interferometer
The optical microfiber interferometer was developed using the optical microfiber taper, which was prepared by flame heating using a butane flame brushing with a heating time of only a few seconds. The single-mode fiber was slowly stretched for two linear stages, and a schematic diagram is shown in Fig. 1(b). The optical microfiber obtained by this method exhibits advantages of a smooth surface and low loss. The diameter of the optical microfiber was 6.6 µm. A scanning electron microscope (SEM) image of the optical microfiber is shown in Fig. 1(c). To facilitate operation, the optical microfiber was folded into a hairpin shape during the experiment. Apart from the negligible effect on the spectrum and sensitivity of the refractive index, the structure of hairpin shape improved the adaptability in the detection of trace biological samples.
2.3 Surface modification and hybridization
The process of the optical microfiber surface functionalization to immobilize the DNA-c molecules is shown in Fig. 1(d). The detailed steps are described as follows:
- (1) The optical microfiber interferometer was soaked in fresh piranha solution for 30 min to obtain enough silicon hydroxyl on the microfiber surface.
- (2) The optical microfiber was rinsed with TE buffer for 10 min to remove the piranha solution remaining on the surface of the optical microfiber. This step was performed between all the followed steps to remove the molecules affecting the immobilization on the surface of the optical microfiber.
- (3) Silicon hydroxyl was allowed to react with 5% APTES for 60 min to silanize the surface of the microfiber. By using atomic force microscopy (AFM, Bioscope Catalyst NanoScope-V), a comparison image of the surface before and after silanization was obtained. The Ra values of the roughness of the bare fiber was 0.47 nm, while that after silanization was 2.69 nm with a Rmax values of 3.20 nm, indicating that a uniform monolayer was formed.
- (4) Then, 2.5% glutaraldehyde solution was allowed to react with the amino group of APTES for 30 min for crosslinking.
- (5) For immobilization, we added 1 µM of DNA-c for 30 min.
- (6) Then, DNA-t hybridization was performed for 30 min. The aminated DNA-c was immobilized on the surface of the optical microfiber to capture the DNA-t molecules with complementary base-pairs.
- (7) Then, DNA-r, which was linked on the surface of Au-NPs, was hybridized with the DNA-t, which led to signal amplification.
To determine the dynamic range of DNA detection, we prepared 10 samples of DNA-t with a 10-fold difference between adjacent samples, with concentrations ranging from 1.0×10−17 M to 1.0×10−8 M. To verify the functionalization of the optical microfiber interferometer that linked the DNA-c and to verify the successful DNA-c hybridization with DNA-t, a fluorescent label was employed. The Olympus (BX53) fluorescence microscope was used at 28 $^\circ \textrm{C}$. A laser source of 490 nm and 550 nm was used for exciting the 6-FAM and the Cy5 fluorophore, respectively. The fluorescent images are shown in Fig. 1(e) to Fig. 1(h). Figure 1(e) shows a faint image of the optical fiber without DNA-c and any fluorescent molecules in the bright field of the fluorescence microscope, whereas Fig. 1(f) showed the same fiber under the excitation, which was almost black. Figure 1(g) shows the DNA-c modified by the 6-FAM fluorophore linked on the fiber surface, and a strong green fluorescence was observed under laser excitation of 490 nm. Similarly, Fig. 1(h) shows red fluorescence under a laser source of 550 nm, which is attributed to the DNA-t modified by the Cy5 fluorophore hybridized with the DNA-c. The fluorescence images demonstrated the actual success of the functionalization procedure.
2.4 Au-NPs preparation and modification with DNA-r
The Au-NPs were synthesized by citrate reduction of HAuCl4 based on a previous study [38,39]. Transmission electron microscopy (TEM, JEOL2100) was performed to analyze the size of the Au-NPs, as shown in Fig. 1(k). The Au-NPs exhibited a spherical structure and exhibited good dispersion; the average particle diameter was approximately 13 nm. As shown in Fig. 1(l), the UV-vis spectrum was collected using a UV-visible spectrophotometer (Cary60) from Agilent Technologies. Curve I in Fig. 1(l) shows that the maximum extinction value of bare Au-NPs is near 520 nm in the UV-vis spectra [40,41], and the absorbance is 0.923. According to the Lambert-Beer's formula [42], the concentration of bare Au-NPs is 3.4 nM. Curve II shows the UV–vis absorption spectra of Au-NPs functionalized by the DNA-r; functionalization was performed based on a previous study [43,44]. The UV-vis absorption spectra of Au-NPs shows that the maximum extinction value moved from 520 nm to 525 nm after functionalized by DNA-r, as shown in curve II in Fig. 1(l), and the wavelength of the absorption peak increased, indicating that the volume of Au-NPs increased. The above result indicates that the Au-NPs were successfully modified by DNA-r. In addition, according to the extinction value [42], the final concentration of bio-functionalized Au-NPs solution was found to be 3.7 nM.
To confirm that DNA-r immobilized on the surface of Au-NPs was successfully hybridized with DNA-t, images obtained from AFM of the microfiber surface after DNA-c hybridization with DNA-t and DNA-t hybridization with DNA-r were collected; the results were shown in Figs. 1(i) and 1(j). Figure 1(i) showed the DNA-c hybridization with DNA-t (before DNA-t hybridization with DNA-r). Figure 1(j) showed DNA-c hybridization with DNA-t and then DNA-t hybridization with DNA-r. The surface roughness and thickness of the microfiber obviously increased after introduction of the Au-NPs, that the maximum roughness value was up to 17.20 nm, and the average roughness value was 6.64 nm. Thus, the DNA-r immobilized on the surface of Au-NPs was successfully hybridized with DNA-t.
2.5 Experimental setup and principle
The optical microfiber interferometer sensor was tested experimentally using the setup illustrated in Fig. 2(a). Light from the broadband source (BBS, 1250 to 1650 nm) was coupled with the optical microfiber interferometer and then propagated along with the optical fiber; An optical spectrum analyzer (OSA, AQ6370D, from YOKOGAWA, operation wavelength ranges from 600 nm to 1700 nm) was used to monitor and record the transmission spectrum in real-time with a resolution of 0.02 nm. When the light propagated along with the optical fiber and then entered the transition region, higher-order modes were excited due to the abrupt change in diameter. When the light propagated to the sensing region, the surface of the optical microfiber produced a strong evanescent field. At the end of the sensing region, the higher-order modes coupled with the optical fiber core and interfered with the fundamental mode [45]. The higher-order modes were sensitive to the RI of the external environment; therefore, the sensitivity of the interferometer can be expressed using Eq. (1) [46,47]:
Fig. 2. (a) Schematic diagram of the optical setup of the optical microfiber biosensor for DNA-t detection. (b) Real-time records of surface modification and hybridization: (I) Rinsing using TE-buffer for 10 min; (II) Silanization using APTES; (III) GD cross-linking; (IV) Immobilization of the amine-modified DNA-c probes; and (V) Hybridization with DNA-t.
After surface functionalization of the optical microfiber interferometer sensor, the DNA-c was immobilized on the surface, as shown in Fig. 1(d). Figure 2(b) shows the spectral responses of the resonant dip throughout the entire functionalization process after hydroxylating with the piranha solution. Generally, the signal response was relatively weak because the variation of RI was very small. However, when the DNA-c captured the DNA-t and was hybridized by complementary base pairing, the RI of the microfiber interferometer surface changed accordingly leading to an obvious signal response. When Au-NPs were functionalized, a large variation in RI was observed. The relative RI variation led to significant changes in the transmission spectrum, thus leading to signal amplification [17]. After binding with DNA-t, the wavelength shifted between the procedures (5) and (6); and the wavelength shifted more obviously after hybridizing signal amplification. Therefore, the concentration of the DNA-t can be calculated according to the relationship between the transmission spectral shift and RI variation.
3. Result and discussion
DNA sequence detection. We fabricated an optical microfiber with DNA-c probe immobilization, and the concentration of complementary specific DNA sequence (DNA-t) ranged from 1.0×10−17 M to 1.0×10−8 M; ten samples were fabricated in total. Experiments without Au-NP signal amplification were performed, and the resonant dip near 1540 nm was selected as the sensing dip. During the process of DNA-c hybridization with different concentrations of the DNA-t, we recorded the real-time wavelength shift, and the kinetic binding curves was shown in Fig. 3(a).
Fig. 3. The kinetic binding curves. (a) Hybridization of DNA-c and DNA-t with different concentrations. (b) Hybridization of DNA-c and DNA-t with different concentrations after introducing Au-NPs.
As the DNA-t concentrations increased from 1.0×10−17 M to 1.0×10−8 M, the transmission spectrum showed a regular shift in Fig. 4(a). The fiber surface was eluted using the piranha solution to etch off the deposited bio-samples on the surface after every experiment. The experiment was performed at least three times independently with the same sensor under the same conditions to further improve the accuracy. As shown in Fig. 4(b), the first reliable signal was obtained at 10 fM. The net wavelength shift showed a good linear relationship with the logarithm of the concentration of DNA-t sequence in the range of 10 fM to 10 nM, with a correlation coefficient of 0.992. The linear regression equation was calculated as y(nm) = 6.842 + 0.457lg(x); the concentration-dependence coefficient was 0.457 nm/lgM.
To promote the performance of the optical microfiber sensor, Au-NPs were bio-functionalized by DNA-r and then used as a signal amplification medium. The procedures of the experiment are similar to those used in previous studies without signal amplification; DNA-t solution concentrations ranged from 1.0×10−17 M to 1.0×10−8 M. During the experiment, 50 μL of Au-NP solution bio-functionalized by DNA-r was added to each concentration of DNA-t solution. And the kinetic curves of the molecular binding was shown in Fig. 3(b). The experiment was performed at least three times independently, and the results are shown in Fig. 5(a). The transmission spectrum showed a significant redshift as the DNA-t concentration increased, and the variation was higher than that without added Au-NP. This is because DNA-r, which was immobilized on the surface of Au- NPs, was hybridized with DNA-t, and then, DNA-c immobilized on the optical microfiber probe captured the DNA-t and hybridized with it. The DNA-c hybridized with DNA-t bound to the Au-NPs, which resulted in a major change in the RI of the fiber surface, resulting in a large shift in the transmission spectrum. The net wavelength shift showed a good linear relationship with the logarithm of concentration; the results are shown in Fig. 5(b). The induced spectral shift with the bulk concentrations can be expressed by y(nm)=10.264+0.663lg(x), and the correlation coefficient is 0.996.
Fig. 4. (a) Measured transmission spectral wavelength shift in the presence of different concentrations of DNA-t (1.0×10−17 M to 1.0×10−8 M). (b) Linear relation with the logarithm of DNA-t concentration ranging from 1.0×10−14 M to 1.0×10−8 M. Each error bar represents the standard deviation of three independent measurements.
Fig. 5. (a) Measured transmission spectrum of DNA-t hybridization at different concentrations (1.0×10−17 M to 1.0×10−8 M) with Au-NPs signal amplification. (b) The linear relationship with the logarithm of DNA-t concentration ranging from 1.0×10−15 M to 1.0×10−8 M with Au-NPs signal amplification. Each error bar represents the standard deviation of three independent measurements.
The temporal evolution of the transmission spectrum was recorded for 60 min at room temperature (28 $^\circ \textrm{C}$), as shown in Fig. 6(a). The transmission spectrum only exhibited irregular fluctuations when the optical microfiber sensor was submerged in TE buffer solution. The standard deviation (σ) of wavelength fluctuations was estimated to be 2.03 pm. By using the σ, the theoretical LOD can be estimated as ${x_{\textrm{LOD}}} = {f^{ - 1}}(\overline y + 3\sigma )$ [49], and the $\overline y$ is the wavelength shift under DNA-t solution with the concentration of the starting point of the linear measurement range. The $\overline y$ without Au-NPs signal amplification was 0.47 nm and that with Au-NPs signal amplification was 0.39 nm. So that the LODs were 11.75 fM without signal amplification and 1.32 fM with that, respectively. The detection capability of the sensor was enhanced after Au-NPs signal amplification. Compared with the former results without signal amplification, the detected initial concentration of the optical microfiber sensor improved by one order of magnitude and the variation of the wavelength shift was at least 50% higher than before.
Fig. 6. (a) Temporal evolution of the transmission spectrum. (b) Measured responses to matched DNA-t and mismatched DNA-t with the same bulk concentration of 10 nM.
Figure 6(b) shows the sensor response for the sequence selectivity testing. Another four target samples were prepared: one base pair mismatched (DNA-t1), three base pair mismatched (DNA-t2), one with all base pairs mismatched (DNA-t3), and a mixture of completely matched and mismatched samples. The selectivity testing experiment was performed under the same conditions, and the samples had the same bulk concentration of 10 nM. In Fig. 6(b), the first column for each sample shows the results without signal amplified by Au-NPs, and the second column shows the result of a signal amplified by Au-NPs. As expected, the sensor recognized the completely matched DNA-t correctly even in the mixed sample, and the transmission spectrum shifted significantly over that of other mismatched samples. With the assistance of Au-NPs, an obvious signal amplification was achieved, as shown for the first and fifth samples in Fig. 6(b). In other mismatched samples, the transmission spectrum did not shift obviously irrespective of signal amplification aided by Au-NPs. The difference in wavelength shift indicated that this optical microfiber sensor can distinguish the specificity DNA-t sequence with only one-base mismatches. The proposed sensor presented good specificity for DNA detection.
Table 2. Comparison of Sensing Performance of Various Label-Free DNA Sensors
Table 2 shows the sensing performances of the proposed sensor in comparison with other biosensors. The optical microfiber interferometer DNA sensor fabricated in this study is convenient to use and is smaller in size than the other sensors. The LOD was also increased compared with other similar optical microfiber interferometer biosensor. Compared with other sensors, the dynamic range and the LOD of this sensor is the highest. To further improve the performance of this sensor, it can be improved by modifying the fiber surface with two-dimensional materials. For example, bimetallic layer MoS2 can be used to enhance fiber surface plasmon resonance (SPR) sensitivity [50] or the optical fiber can be coated with a Au/WS2 multilayer film to improve the sensitivity [51]. The proposed sensor has a good specificity and large range of line measurements, which is also important in some fields such as forensic science.
4. Conclusion
In this study, a quantitative detection approach for DNA sequences using an optical microfiber and using Au-NPs for signal amplification is proposed and demonstrated experimentally. The transmission spectrum showed a small shift when the optical microfiber functionalized with DNA-c was bound with the complementary DNA-t, leading to a change in the RI. After signal amplification using Au-NPs bio-functionalized by DNA-r, an enhanced shift was observed only in the case of completely complementary DNA-t. The results showed the LOD could reach up to 1.32 fM, and the concentration range from 1 fM to 10 nM showed a good logarithmic-linear response. This optical microfiber interferometer sensor showed high specificity (single-base level). Other recognition elements can be applied as target analytes in our sensor, such as proteins or contaminants, using the same strategy as described, thus making this technology suitable for powerful and versatile bio-sensing platforms.
Funding
National Natural Science Foundation of China (U1701268, 61705083, 21807042); Natural Science Foundation of Guangdong Province (2019A1515011144, 2018A030313325); Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02X105); Tip-top Scientific and Technical Innovative Youth Talents of Guangdong Special Support Program (2019TQ05X136); Guangzhou Municipal Science and Technology Project (201904020032); Fundamental Research Funds for the Central Universities.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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